Chemical amplification based on fluid partitioning in an immiscible liquid

ABSTRACT

A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

This application is a Reissue of application Ser. No. 10/389,130, filedMar. 14, 2003, issued as U.S. Pat. No. 7,041,481 on May 9, 2006.

The United States Government has rights in this invention pursuant toContract No. W-7405-ENG-48 between the United States Department ofEnergy and the University of California for the operation of LawrenceLivermore National Laboratory.

BACKGROUND

1. Field of Endeavor

The present invention relates to chemical amplification and moreparticularly to chemical amplification based on fluid partitioning.

2. State of Technology

U.S. Pat. No. 4,683,202 issued Jul. 28, 1987; U.S. Pat. No. 4,683,195issued Jul. 28, 1987; and U.S. Pat. No. 4,800,159 issued Jan. 24, 1989to Kary B. Mullis et al provide background information. The patentsdescribe processes for producing any particular nucleic acid sequencefrom a given sequence of DNA or RNA in amounts which are large comparedto the amount initially present. The DNA or RNA may besingle-or-double-stranded, and may be a relatively pure species or acomponent of a mixture of nucleic acids. The process utilizes arepetitive reaction to accomplish the amplification of the desirednucleic acid sequence. The extension product of one primer whenhybridized to the other becomes a template for the production of thedesired specific nucleic acid sequence, and vice versa, and the processis repeated as often as is necessary to produce the desired amount ofthe sequence.

U.S. Pat. No. 6,503,715 for a nucleic acid ligand diagnostic biochipissued Jan. 7, 2003 provides the following background information,“Methods are provided in the instant invention for obtaining diagnosticand prognostic Nucleic acid ligands, attaching said ligands to aBiochip, and detecting binding of target molecules in a Bodily to saidBiochip-bound Nucleic acid ligands.” In one embodiment of the instantinvention, one or more Nucleic acid ligands are chosen that bind tomolecules known to be diagnostic or prognostic of a disease; theseligands are then attached to the Biochip. Particular methods forattaching the Nucleic acid ligands to the Biochip are described below inthe section entitled “Fabrication of the Nucleic Acid Biochip.” TheBiochip may comprise either (i) Nucleic acid ligands selected against asingle target molecule; or more preferably, (ii) Nucleic acid ligandsselected against multiple target molecules.

U.S. Patent Application No. 2002/0197623 for nucleic acid detectionassays published Dec. 26, 2002 provides the following backgroundinformation, “means for the detection and characterization of nucleicacid sequences, as well as variations in nucleic acid sequences . . .methods for forming a nucleic acid cleavage structure on a targetsequence and cleaving the nucleic acid cleavage structure in asite-specific manner. The structure-specific nuclease activity of avariety of enzymes is used to cleave the target-dependent cleavagestructure, thereby indicating the presence of specific nucleic acidsequences or specific variations thereof.”

SUMMARY

Features and advantages of the present invention will become apparentfrom the following description. Applicants are providing thisdescription, which includes drawings and examples of specificembodiments, to give a broad representation of the invention. Variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those skilled in the art from this descriptionand by practice of the invention. The scope of the invention is notintended to be limited to the particular forms disclosed and theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

The present invention provides an apparatus for nucleic acidamplification of a sample comprising means for partitioning the sampleinto partitioned sections and means for performing PCR on thepartitioned sections of the sample. Another embodiment of the inventionprovides an apparatus for nucleic acid amplification and detection of asample comprising means for partitioning the sample into partitionedsections, means for performing PCR on the partitioned sections of thesample, and means for detection and analysis of the partitioned sectionsof the sample. The present invention also provides a method of nucleicacid amplification of a sample comprising the steps of partitioning thesample into partitioned sections and subjecting the partitioned sectionsof the sample to PCR. Another embodiment of a method of the presentinvention provides a method of nucleic acid amplification and detectionof a sample comprising the steps of partitioning the sample intopartitioned sections, subjecting the partitioned sections of the sampleto PCR, and detecting and analyzing the partitioned sections of thesample.

The invention is susceptible to modifications and alternative forms.Specific embodiments are shown by way of example. It is to be understoodthat the invention is not limited to the particular forms disclosed. Theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of the specification, illustrate specific embodiments of theinvention and, together with the general description of the inventiongiven above, and the detailed description of the specific embodiments,serve to explain the principles of the invention.

FIG. 1 is a flow diagram illustrating one embodiment of a systemconstructed in accordance with the present invention.

FIG. 2 is a flow diagram illustrating another embodiment of a systemconstructed in accordance with the present invention.

FIG. 3 is a diagram of another embodiment of a system constructed inaccordance with the present invention.

FIG. 4 is a diagram of another embodiment of a system constructed inaccordance with the present invention.

FIG. 5 is a diagram of another embodiment of a system constructed inaccordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, to the following detailed description,and to incorporated materials; detailed information about the inventionis provided including the description of specific embodiments. Thedetailed description serves to explain the principles of the invention.The invention is susceptible to modifications and alternative forms. Theinvention is not limited to the particular forms disclosed. Theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

Referring now to the drawings, and in particular to FIG. 1, a flowdiagram of one embodiment of a system constructed in accordance wish thepresent invention is illustrated. The system is designated generally bythe reference numeral 100. The system 100 provides a method andapparatus for performing extremely rapid nucleic acid amplification. Theflow diagram illustrating system 100 shows block 101 “partitioning” thesample and block 102 performing “CR” on the sample. The system 100provides an apparatus for nucleic acid amplification of a samplecomprising means for partitioning the sample and means for performingPCR on the sample. The system 100 also provides a method of nucleic acidamplification of a sample comprising the steps of partitioning thesample and subjecting the sample to PCR. The system 100 has applicationwherever current PCR-type systems exist.

In block 101 a chemical reagent and an input sample are “partitioned”into a large number of microdroplets or other forms of fluid partitionsprior to amplification in block 102. The partitioning 101 involvesdispersing the DNA-containing solution. For example the partitioning 101can be accomplished by dispersing the DNA-containing solution in animmiscible carrier liquid. The DNA-containing solution is dispersed inthe immiscible carrier fluid as microdroplets. The DNA-containingsolution can be partitioned in other ways, for example, by beingdispersed as liquid slugs separated by the carrier fluid, as an emulsionwith the carrier fluid, or by using a gelling agent that preventstransfer of DNA between partitioned regions. The DNA-containing solutioncan also be partitioned mechanically by partitioning the fluid intomicro-tubes or capillaries, or into micro-wells.

With the system 100, each partitioned DNA-containing fluid volumecontains the necessary biochemical constituents for selectivelyamplifying a specified portion of a sample DNA via polymerase chainreaction (PCR). The target DNA can be detected by monitoring for thecolorimetric indicator (e.g., flourescence or optical absorption)generated with each DNA template duplication sequence.

In block 102 selected portions of each nucleic acid sample are amplifiedusing polymerase chain reaction (PCR), with the product contained ineach partitioned fluid volume. This results in much more concentratedamplification product, since the volume containing the reaction is sosmall.

The polymerase chain reaction (PCR), is a cyclic process whereby a largequantity of identical DNA strands can be produced from one originaltemplate. The procedure was developed in 1985 by Kerry Mullis, who wasawarded the 1993 Nobel prize in chemistry for his work. In PCR, DNA isimmersed in a solution containing the enzyme DNA polymerase, unattachednucleotide bases, and primers, which are short sequences of nucleotidesdesigned to bind with an end of the desired DNA segment. Two primers areused in the process: one primer binds at one end of the desired segmenton one of the two paired DNA strands, and the other primer binds at theopposite end on the other strand. The solution is heated to break thebonds between the strands of the DNA, then when the solution cools, theprimers bind to the separated strands, and DNA polymerase quickly buildsa new strand by joining the free nucleotide bases to the primers in the5′-3′ direction. When this process is repeated, a strand that was formedwith one primer binds to the other primer, resulting in a new strandthat is restricted solely to the desired segment. Thus the region of DNAbetween the primers is selectively replicated. Further repetitions ofthe process can produce a geometric increase in the number of copies,(theoretically 2n if 100% efficient whereby n equals the number ofcycles), in effect billions of copies of a small piece of DNA can bereplicated in several hours.

A PCR reaction is comprised of (a) a double-stranded DNA molecule, whichis the “template” that contains the sequence to be amplified, (b)primer(s), which is a single-stranded DNA molecule that can anneal(bind) to a complimentary DNA sequence in the template DNA; (c) dNTPs,which is a mixture of dATP, dTTP, dGTP, and dCTP which are (henucleotide subunits that will be put together to form new DNA moleculesin the PCR amplification procedure; and (d) Taq DNA polymerase, theenzyme which synthesizes the new DNA molecules using dNTPs.

Current amplification systems are limited in practice to half hour typeamplification and detection windows (−30 cycles, 1 minute/cycle). Thesystem 100 provides faster amplification. This has many applications,foe example, in Homeland Defense applications, faster detection methods(a few minutes) can push the deployment of these sensors from “detect totreat” to “detect to protect,” having a serious impact on the number ofcasualties from a massive bioagent release.

The system 100 has significant advantages over typical bulk DNAdetection techniques (even microscale bulk solution approaches),including (1) much faster detection time through a reduction in thetotal number of temperature cycles required, (2) a reduction in the timefor each cycle, and (3) removing interference from competing DNAtemplates. The system 100 achieves a reduction in the total number ofcycles by limiting the dilution of the optically generated signal (e.g.,fluorescence or absorption). The formation of partitioned fluid volumesof the DNA-containing solution effectively isolates the fluid volumeswhich contain the target DNA from the fluid volumes that do not containthe target DNA. Therefore, the dilution of the optical signal is largelyeliminated, allowing much earlier detection. This effect is directlyrelated to the number of fluid partitions formed from the initialsample/reagent pool.

The system 100 achieves a reduction in the total number of cycles thatare needed by limiting the dilution of the optically generated signal(e.g., fluorescence or absorption). The formation of partitioned fluidvolumes of the DNA-containing solution effectively isolates the fluidvolumes which contain the target DNA from the fluid volumes that do notcontain the target DNA. Therefore, the dilution of the optical signal islargely eliminated, allowing much earlier detection. This effect isdirectly related to the number of fluid partitions formed from theinitial sample/reagent pool. The effect of the number of fluidpartitions on the number of cycles required for detection can bedescribed by the following Equation E1:$N = \frac{1{n\lbrack {D_{L}{A_{N}( \frac{V}{X} )}} \rbrack}}{1{n(2)}}$where: N=number of cycles; D_(L,)=detection limit for optical signal[moles/liter]; X=initial number of DNA molecules; V=volume containingDNA molecules [liters]; A_(N)=Avagadro's number [6.023×1023molecules/mole]. From Equation E1 it is clear that N, the number ofcycles until detection, decreases as V, the partitioned fluid volume,decreases.

The system 100 reduces the duration of each temperature cycle byeffectively increasing the concentration of reactants by enclosing themin picoliter type volumes. Since reaction rates depend on theconcentration of the reactants, the efficiency of a partitioned fluidvolume or droplet should be higher than in an ordinary vessel (such as atest tube) where the reactant quantity (DNA quantity) is extremely low.It is estimated that through the reduction in the number of cycles andthe reduction in the time required for each cycles that the FPDDtechnique can reduce the detection time by an order of magnitude ascompared to bulk solution DNA detection techniques.

The system 100 facilitates removal of interference from competing DNAtemplates. Given the extremely small volumes involved withFluid-Partitioned DNA Detection (FPDD), it is possible to isolate asingle template of the target DNA in a given partitioned volume ormicrodroplet. For example, the formation of 2000 partitioned fluidvolumes or microdroplets (each with a volume of 5×10′9 5×10⁻⁹ liters)made by dividing a bulk solution of 10 microliters containing 200 2000DNA molecules, would result in one DNA molecule per microdroplet onaverage. This makes it possible to amplify only one template in mixturescontaining many kinds of templates without interference. This isextremely important in processing of real world aerosol samplescontaining complex mixtures of DNA from many sources, and has directapplication in screening of cDNA libraries.

Referring now to FIG. 2, a flow diagram of another embodiment of asystem constructed in accordance with the present invention isillustrated. The system is designated generally by the reference numeral200. The How diagram illustrating system 200 shows block 201“partitioning” the sample, block 202 performing “PCR” on the sample, andblock 203 “detection and analysis.” The system 200 provides a method andapparatus for performing extremely rapid nucleic acid amplification anddetection. The system 200 provides an apparatus for nucleic acidamplification of a sample comprising means for partitioning the sampleinto partitioned sections, means for performing PCR on the partitionedsections, and means for detection and analysis of the partitionedsections. The system 200 also provides a method of nucleic acidamplification of a sample comprising the steps of partitioning thesample into partitioned sections, subjecting the partitioned sections toPCR, and detecting and analyzing the partitioned sections of the sample.

In block 201 a chemical reagent and an input sample are “partitioned”into a large number of microdroplets or other forms of fluid partitionsprior to amplification. The system 200 achieves a reduction in the totalnumber of cycles by limiting the dilution of the optically generatedsignal (e.g., fluorescence or absorption). The formation of partitionedfluid volumes of the DNA-containing solution effectively isolates thefluid volumes which contain the target DNA from the fluid volumes thatdo not contain the target DNA. Therefore, the dilution of the opticalsignal is largely eliminated, allowing much earlier detection. Thiseffect is directly related to the number of fluid partitions formed fromthe initial sample/reagent pool.

In block 202 selected portions of each nucleic acid sample are thenamplified using polymerase chain reaction (PCR), with the productcontained in each partitioned fluid volume. This results in much moreconcentrated amplification product, since the volume containing thereaction is so small. If a Taqman type detection approach is used,fluorescent dye molecules unquenched by the PCF amplification are alsomore concentrated, making possible earlier optical based detection.Since it is possible to contain very amounts of the starting target DNAin each partition fluid volume, inhibitory competition fromnear-neighbor DNA templates is less allowing screening of very dilutesamples.

In block 203 partitioned portions of the sample are detected bymonitoring for the colorimetric indicator (e.g., fluorescence or opticalabsorption) generated with each DNA template duplication sequence. Thepartitioned portions of the sample are optically probed to detect thecolorimetric indicator which signals the presence of the target DNA. Thepartitioned portions of the sample can also be scanned optically todetect the colorimetric indicator signaling the presence of the targetDNA. In one embodiment, fluorescence, generated by degradation of thedye/quencher pair on the primer, is detected using a confocal imagingsystem such as that employed in conventional flow cytometers. Scatteringprofiles from individual microdroplets, as in conventional flowcytometers, can be used to eliminate background signal from otherparticles.

The system 200 has application wherever current PCR-type systems exist,including medical, drug-discovery, biowarfare detection, and otherrelated fields. Biowarfare detection applications include identifying,detecting, and monitoring bio-threat agents that contain nucleic acidsignatures, such as spores, bacteria, etc. Biomedical applicationsinclude tracking, identifying, and monitoring outbreaks of infectiousdisease. The system 200 provides rapid, high throughput detection ofbiological pathogens (viruses, bacteria, DNA in biological fluids,blood, saliva, etc.) for medical applications. Forensic applicationsinclude rapid, high throughput detection of DNA in biological fluids forforensic purposes. Food and beverage safety applications includeautomated food testing for bacterial contamination.

Referring now to FIG. 3, a diagram of another embodiment of a systemconstructed in accordance with the present invention is illustrated. Thesystem is designated generally by the reference numeral 300. The system300 provides an instrument for performing Fluid-Partitioned DNADetection (FPDD) with PCR based detection and amplification. The system300 includes a partitioning section 301, a PCR section 302, and adetection and analysis section 303.

The partitioning section 301 includes a sample introduction unit 304 anda unit 305 where the sample and a PCR reagent are combined. The sampleand a PCR reagent are injected through a small orifice 306. Theinjection of the sample through the small orifice 306 producesmicrodroplets 308.

The PCR section 302 includes a continuous tube 309 for circulating themicrodroplets 308 and suspended in an immiscible carrier fluid 314. Themicrodroplets 308 suspended in an immiscible carrier fluid 314 arepumped through the continuous tube 309 by pump 311. The microdroplets308 suspended in an immiscible carrier fluid 314 are cycled throughheater 310 and cooler 315 to perform PCR.

The detection and analysis section 303 includes a blue laser 312 and adetector 313. The laser 312 is projected upon the droplets 308 as theypass through tube 308 between the laser 312 and the detector 313.

In the system 300, the DNA-containing solution is partitioned into manymicrodroplets 308 and suspended in an immiscible carrier fluid 314. Themicrodroplets 308 are formed by forcing the PCR mix (sample and reagent)through the small orifice or microjet 306. These microdroplets 308 arethen captured in the immiscible fluid 314, such as mineral oil, andflowed past the healing element 310 and cooler 315. An optical signal(e.g., fluorescence or optical absorption), generated by degradation ofthe dye/quencher pair on the primer, is detected rising a confocalimaging system such as that employed in conventional flow cytometers.Scattering profiles from individual microdroplets, as in conventionalflow cytometers, can be used to eliminate background signal from otherparticles. Once exposed to multiple heating cycles, the microdropletscan be identified and probed for an optical signal at rates of severalthousand per second.

The FPDD system achieves a reduction in the total number of cycles bylimiting the dilution of the optically generated signal (e.g.,fluorescence or absorption). The formation of partitioned fluid volumesof the DNA-containing solution effectively isolates the fluid volumeswhich contain the target DNA from the fluid volumes that do not containthe target DNA. Therefore, the dilution of the optical signal is largelyeliminated, allowing much earlier detection. This effect is directlyrelated to the number of fluid partitions formed from the initialsample/reagent pool. The effect of the number of fluid partitions on thenumber of cycles required for detection is described by the Equation E1set out earlier.

The FPDD technique reduces the duration of each temperature cycle byeffectively increasing the concentration of reactants by enclosing themin picoliter type volumes. Since reaction rates depend on theconcentration of the reactants, the efficiency of a partitioned fluidvolume or droplet should be higher than in an ordinary vessel (such as atest tube) where the reactant quantity (DNA quantity) is extremely low.It is estimated that through the reduction in the number of cycles andthe reduction in the time required for each cycles that the FPDDtechnique can reduce the detection time by an order of magnitude ascompared to bulk solution DNA detection techniques

The FPDD technique facilitates removal of interference from competingDNA templates. Given the extremely small volumes involved with FPDD, itis possible to isolate a single template of the target DNA in a givenpartitioned volume or microdroplet. For example, the formation of 2000partitioned fluid volumes or microdroplets (each with a volume of 5×10⁻⁹liters) made by dividing a bulk solution of 10 microliters containing200 2000 DNA molecules, would result in one DNA molecule permicrodroplet on average. This makes it possible to amplify only onetemplate in mixtures containing many kinds of templates withoutinterference. This is extremely important in processing of real worldaerosol samples containing complex mixtures of DNA from many sources,and has direct application in screening of cDNA libraries.

With this new bioassay technique, each partitioned DNA-containing fluidvolume contains the necessary biochemical constituents for selectivelyamplifying a specified portion of a sample DNA via polymerase chainreaction (PCR). The target DNA is detected by monitoring for thecolorimetric indicator (e.g., fluorescence or optical absorption)generated with each DNA template duplication sequence.

The system 300 provides a fast, flexible and inexpensive highthroughput, bioassay technology based on creation and suspension ofmicrodroplets in an immiscible carrier stream. Each microdropletcontains the necessary biochemical constituents for selectivelyamplifying and fluorescently detecting a specified portion of a sampleDNA via polymerase chain reaction (PCR). Once exposed to multipleheating cooling cycles, the microdroplets can be identified and probedfor fluorescent signal at rates of several thousand per second.

Isolating the PCR reaction in such small (picoliter) volumes provides anorder of magnitude reduction in overall detection time by:

-   -   (1) reducing the duration of each temperature cycle—the        concentration of reactants increases by enclosing them in        picoliter type volumes. Since reaction kinetics depend on the        concentration of the reactant, the efficiency of a microdroplet        should be higher than in an ordinary vessel (such a test tube)        where the reactant quantity is infinitesimal    -   (2) reducing the total number of cycles—dilution of the        fluorescently generated signal is largely eliminated in such a        small volume, allowing much earlier detection. This effect is        directly related to the number of microdroplets formed from the        initial sample/reagent pool. Since PCR is an exponential        process, for example, 1000 microdroplets would produce a signal        10 cycles faster than typical processing with bulk solutions.    -   (3) removing interference from competing DNA templates—given the        extremely small volumes involved, it is possible to isolate a        single template of the target DNA in a given microdroplet. A pL        microdoplet filled with a 1 pM solution, for example, will be        occupied by only one molecule on average. This makes it possible        to amplify only one template in mixtures containing many kinds        of templates without interference. This is extremely important        in processing of real world aerosol samples containing complex        mixtures of DNA from many sources, and has direct application in        screening of precious cDNA libraries.

Referring now to FIG. 4, an illustration of another embodiment of asystem constructed in accordance with the present invention isillustrated. The system is designated generally by the reference numeral400. The system 300 provides system for nucleic acid amplification of asample. The system 400 includes means for partitioning the sample intopartitioned sections and means for performing PCR on the partitionedsections of the sample.

The sample is separated into immiscible slugs 406, 407, and 408. Theimmiscible slugs 406, 407, and 408 are formed through a system ofmicrofluidics. Background information on microfluidics is contained inU.S. Pat. No. 5,876,187 for micropumps with fixed valves to Fred K.Forster et al., patented Mar. 2, 1999. As stated in U.S. Pat. No.5,876,187, “Miniature pumps, hereafter referred to as micropumps, can beconstructed using fabrication techniques adapted from those applied tointegrated circuits. Such fabrication techniques are often referred toas micromachining. Micropumps are in great demand for environmental,biomedical, medical, biotechnical, printing, analytical instrumentation,and miniature cooling applications.” Microchannels 403, 404, and 405 areformed in substrates 401 and 402. The disclosures of U.S. Pat. Nos.5,876,187 and 5,876,187 are incorporated herein by reference.

The immiscible slugs 406, 407, and 408 can be moved through themicrochannels using magnetohydrodynamics. Background information onmagnetohydrodynamics is contained in U.S. Pat. No. 6,146,103 formicromachined magnetohydrodynamic actuators and sensors to Abraham P.Lee and Asuncion V. Lemoff, patented Nov. 14, 2000. As stated in U.S.Pat. No. 6,146,103, “Microfluidics is the field for manipulating fluidsamples and reagents in minute quantities, such as in micromachinedchannels, to enable handheld bioinstrumentation and diagnostic toolswith quicker process speeds. The ultimate goal is to integrate pumping,valving, mixing, reaction, and detection on a chip for biotechnological,chemical, environmental, and health care applications. Most micropumpsdeveloped thus far have been complicated, both in fabrication anddesign, and often are difficult to reduce in size, negating manyintegrated fluidic applications. Most pumps have a moving component toindirectly pump the fluid, generating pulsatile flow instead ofcontinuous flow. With moving parts involved, dead volume is often aserious problem, causing cross-contamination in biological sensitiveprocesses. The present invention utilizes MHDs for microfluid propulsionand fluid sensing, the microfabrication methods for such a pump, and theintegration of multiple pumps for a microfluidic system. MHDs is theapplication of Lorentz force law on fluids to propel or pump fluids.Under the Lorentz force law, charged particles moving in a uniformmagnetic field feel a force perpendicular to both the motion and themagnetic field. It has thus been recognized that in the microscale, theMHD forces are substantial for propulsion of fluids throughmicrochannels as actuators, such as a micropump, micromixer, ormicrovalve, or as sensors, such as a microflow meter, or viscositymeter. This advantageous scaling phenomenon also lends itself tomicromachining by integrating microchannels with micro-electrodes.” Thedisclosure of U.S. Pat. No. 6,146,103 is incorporated herein byreference.

The means for performing PCR on the partitioned sections of the samplecan be a system for alternately heating and cooling the immiscible slugs406, 407, and 408. Alternatively, the means for performing PCR on thepartitioned sections of the sample can be a system for alternatelyheating and cooling the immiscible slugs 406, 407, and 408 can be asystem for moving the immiscible slugs 406, 407, and 408 through zonesfor heating and cooling. An example of such a system is shown in U.S.patent application No. 2002/0127152 published Sep. 12, 2002 for aconnectively driven PCR thermal-cycling system described as follows: “Apolymerase chain reaction system provides an upper temperature zone anda lower temperature zone in a fluid sample. Channels set up convectioncells in the fluid sample and move the fluid sample repeatedly throughthe upper and lower temperature zone creating thermal cycling.” Thedisclosure of U.S. Patent Application No. 2002/0127152 is incorporatedherein by reference.

In another embodiment of the invention, the DNA-containing solution ispartitioned by adding a gelling agent to the solution to form cells ofpartitioned volumes of fluid separated by the gelling agent. Using thisapproach for fluid partitioning, the DNA-containing solution is gelledin a tube or as a very thin layer. For example, it can be in a thinlayer between flat plates and the surface of the thin film can beoptically probed spatially in directions parallel to the film surface todetect micro-regions in the film where the colorimetric indicatorsuggests the presence of the target DNA.

Another embodiment of the invention is to partition the DNA-containingsolution as microdroplets in an immiscible fluid where the droplets arearranged in a two-dimensional array such that the array of microdropletscan be optically probed to detect the colorimetric indicator whichsignals the presence of the target DNA. In this approach a solidhydrophobic substrate supports the microdroplets. For example, in smallindentations, and the immiscible “partitioning” fluid is less dense thanthe aqueous DNA-containing solution.

In another embodiment of the invention the DNA-containing solution ispartitioned using mechanical means. For example, the DNA-containingsolution can be partitioned into an array of capillaries, microtubes, orwells. In this approach, the micro vessels holding each partitionedfluid volume can be scanned optically to detect the colorimetricindicator signaling the presence of the target DNA.

Referring now to FIGS. 5A, 5B, and 5C example representations of themechanical partitioning approach for DNA detection using fluidpartitioning are shown. In FIG. 5A a line of capillaries or micro-tubes501 are used for partitioning and holding the DNA containing solution.In FIG. 5B an array 502 of capillaries or micro-tubes are used forpartitioning the DNA-containing solution. In FIG. 5C a micro-wells ormicro-vessels unit 503 is used for partitioning and holding theDNA-containing solution.

While the invention may be susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and have been described in detail herein.However, it should be understood that the invention is not intended tobe limited to the particular forms disclosed. Rather, the invention isto cover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the followingappended claims.

1. An apparatus for nucleic acid amplification of a sample, comprising:means for partitioning said sample into partitioned sections, whereinsaid means for partitioning said sample into partitioned sectionscomprises an injection orifice, and means for performing PCR on saidpartitioned sections of said sample.
 2. The apparatus for nucleic acidamplification of a sample of claim 1 wherein said injection orifice isan injection orifice that produces microdroplets.
 3. The apparatus fornucleic acid amplification of a sample of claim 1 wherein said injectionorifice is an injection orifice that injects said sample and a PCRreagent.
 4. The apparatus for nucleic acid amplification of a sample ofclaim 1 wherein said means for performing PCR on said partitionedsections of said sample comprises a continuous tube for circulating saidpartitioned sections of said sample through a heater to perform PCR. 5.The apparatus for nucleic acid amplification of a sample of claim 1wherein said means for performing PCR on said partitioned sections ofsaid sample comprises a continuous tube for circulating said partitionedsections of said sample through a heater and cooler to perform PCR. 6.The apparatus for nucleic acid amplification of a sample of claim 1wherein said means for performing PCR on said partitioned sections ofsaid sample comprises a pump, a continuous tube, and a heater.
 7. Theapparatus for nucleic acid amplification of a sample of claim 1including means for detection and analysis of said partitioned sectionsof said sample comprising a laser and a detector.
 8. The apparatus fornucleic acid amplification of a sample of claim 1 including means fordetection and analysis of said partitioned sections of said samplecomprising a blue laser and a detector.
 9. The apparatus for nucleicacid amplification of a sample of claim 1 wherein said means forpartitioning said sample into partitioned sections comprises means forseparating said sample into immiscible slugs.
 10. A method of nucleicacid amplification of a sample, comprising the steps of: partitioningsaid sample into partitioned sections, wherein said step of partitioningsaid sample into partitioned sections comprises flowing said samplethrough an injection orifice into an immiscible carrier fluid, andsubjecting said partitioned sections of said sample to PCR.
 11. Themethod of claim 10 wherein the nucleic acid amplification of a samplecomprises PCR amplification of a DNA target.
 12. The method of claim 11wherein said partitioned sections contain, on average, a single templateof a DNA target, and wherein said single template is amplified withinsaid partitioned sections.
 13. The method of claim 12 wherein saidsample comprises multiple DNA targets, and wherein multiple partitionedsections have a single template of a different DNA target such that saidsingle template is amplified within said multiple partitioned sections.14. The method of claim 10, wherein the partitioned sections are passedby a detector to detect the amount of amplification.
 15. The method ofclaim 14 wherein the detector is a light detector.
 16. The method ofclaim 15 wherein an amount of amplification is indicated byfluorescence.
 17. The method of claim 16 where a fluorophore dye isused.
 18. The method of claim 15 wherein a laser is projected upon thepartitioned sections as they pass between the laser and detector. 19.The method of claim 15 wherein the detector comprises a confocal imagingsystem.
 20. The method of claim 15 wherein scattering profiles from thepartitioned sections are used to eliminate background signals.
 21. Themethod of claim 16 wherein the partitioned sections are probed forfluorescent signal at a rate of several thousand per second.
 22. Anucleic acid amplification apparatus comprising a microdroplet generatorcomprising an orifice, wherein said orifice connects a sample flowpathway to a channel or tube comprising an immiscible fluid, and whereinsaid channel or tube passes through a heating element.
 23. The apparatusof claim 22 further comprising a cooler.
 24. The apparatus of claim 22wherein said microdroplet generator is capable of producingmicrodroplets with volumes in the picoliter range.
 25. The apparatus ofclaim 22 wherein said microdroplet generator is capable of producingmicrodroplets having volumes of about 5×10 ⁻⁹ liters to 1×10 ⁻¹² liters.26. The apparatus of claim 22 wherein the immiscible fluid is mineraloil.
 27. The apparatus of claim 22, further comprising a a pump formoving generated microdroplets in said immiscible fluid through thechannel or tube.
 28. The apparatus of claim 27 further comprising a pumpfor moving the microdroplets through the channel or tube.
 29. Theapparatus of claim 27 wherein the tube is a continuous tube.
 30. Theapparatus of claim 27 wherein the channel is a micromachined channel.31. The apparatus of claim 28 wherein the pump for moving themicrodroplets comprises a magnetohydrodynamic (MHD) element.
 32. Theapparatus of claim 27 wherein the channel or tube is heated and cooled.33. The apparatus of claim 27 wherein the channel or tube extendsthrough a heater and a cooler.
 34. A nucleic acid amplificationapparatus comprising: a microdroplet generator comprising an orificewherein said orifice connects a sample flow pathway to a channel or tubecomprising an immiscible fluid, wherein said channel or tube passesthrough a heating element; and wherein said apparatus further comprisesa detector capable of detecting microdroplets in said immiscible fluid.35. The apparatus of claim 34 wherein the detector is positioned suchthat generated microdroplets suspended in said immiscible fluid pass bythe detector as they are moved through the channel or tube.
 36. A methodfor nucleic acid amplification comprising: producing microdropletswithin an immiscible fluid in a channel or tube; wherein themicrodroplets comprise nucleic acids and components for performingnucleic acid amplification; moving the microdroplets through the channelor tube; and thermal cycling the microdroplets in the channel or tube toamplify the nucleic acids.
 37. The method of claim 36 wherein thenucleic acid amplification comprises PCR.
 38. The method of claim 36wherein the thermal cycling of the microdroplets comprises passing themicrodroplets through a heater and a cooler.
 39. The method of claim 36wherein the thermal cycling of the microdroplets comprises heating andcooling the channel or tube comprising the microdroplets.
 40. The methodof claim 36 further comprising passing the microdroplets by a detectorto detect an amount of amplification.
 41. The method of claim 40 whereinthe detector is a light detector.
 42. The method of claim 41 wherein theamount of amplification is indicated by fluorescence.
 43. The method ofclaim 42 where a fluorophore dye is used.
 44. The method of claim 41wherein a laser is projected upon the microdroplets as they pass betweenthe laser and detector.
 45. The method of claim 41 wherein the detectorcomprises a confocal imaging system.
 46. The method of claim 41 whereinscattering profiles from the microdroplets are used to eliminatebackground signals.
 47. A method comprising: diluting a samplecomprising a plurality of DNA targets and PCR reagents; partitioning thesample into microdroplets in an immiscible fluid in a tube or channel ofa microfluidic device, wherein a plurality of microdroplets containing asingle template of the target DNA are formed; and amplifying the targetDNA in the microdroplets by heating and cooling such that a plurality ofsingle templates within the microdroplets are amplified.
 48. A methodcomprising: a. performing PCR on a microdroplet suspended in animmiscible fluid in a microchannel, wherein said PCR comprises aplurality of cycles; b. passing said microdroplet through saidmicrochannel past a detector; and c. detecting a PCR amplificationproduct in said microdroplet.
 49. The method of claim 48, wherein saidmicrodroplet is isolated from a bulk solution, and whereby the number ofPCR cycles needed to detect said amplication product in saidmicrodroplet is less than the number of PCR cycles needed to detectamplication product in said bulk solution.
 50. The method of claim 48,wherein said microdroplet is isolated from a bulk solution, and wherebythe time needed for each cycle of PCR on said microdroplet is less thanthe time needed for each cycle of PCR in said bulk solution.
 51. Themethod of claim 48 wherein the volume of said microdroplet is about 5×10⁻⁹ liters to 1×10 ⁻¹² liters.